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SBNO1 Is an Epigenetic Driver with Common and Distinct Roles in HCC and CCA
1
Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany; 2 Medical Research Centre, University of Heidelberg, Mannheim, Germany; 3 Department of Neuropathology, University Hospital Heidelberg and Clinical Cooperation Unit Neuropathology, German Cancer Research Center, Heidelberg, Germany; 4 Department of General Visceral and Transplantation Surgery, University Hospital Heidelberg, Heidelberg, Germany; 5 Institute of Pathology and Neuropathology, Hospital RKH Kliniken Ludwigsburg, Ludwigsburg, Germany; 6Institute of Tissue Medicine and Pathology, University of Bern, Bern, Switzerland.
Background: The Notch pathway is an evolutionary conserved signaling pathway playing a pivotal role in appropriate
liver development and regeneration. Interestingly, aberrant Notch signaling is a potential driver of liver inflammation,
formation and progression of hepatocellular (HCC) and cholangiocarcinoma (CCA). However, little is known regarding
major up- and downstream components of the Notch pathway and their relevance in liver cancer.
Aims: Genetic studies in Drosophila showed that the knockout of strawberry notch (sno) mimics loss of Notch suggesting
that sno is a component of the Notch signaling pathway. Here, we aimed to elucidate the role of the mammalian
Strawberry Notch 1 (SBNO1) protein in HCC and CCA development.
Methods: Publicly available HCC and CCA gene expression and proteomics data sets were analyzed for SBNO1 mRNA
and protein expression and association with patient outcome. Tissue microarrays of CCA and non-neoplastic tissue were
immunohistochemically evaluated for SBNO1 protein expression. SBNO1 was downregulated using siRNA or sgRNA in
HCC cell lines followed by cell viability, colony formation and migration assays and gene expression analysis by RNA
sequencing. To identify SBNO1 protein interaction partners, SBNO1 protein was fused to BirA and BioID labeling was
performed in living tumor cells. SBNO1 function was evaluated in a syngeneic mouse model using Hep55.1C cell line and
three different liver tumor models of hydrodynamic tail vein injection in wildtype mice. Murine tissues were evaluated
by immunohistochemical staining.
Results: We found SBNO1 protein but not mRNA to be significantly increased in HCC, intrahepatic (iCCA) and perihilar
(pCCA) but not distal CCA (dCCA). SBNO1 protein localized to the nucleus of HCC and CCA tumor cells suggesting a role in
gene regulation. Inhibition of SBNO1 expression resulted in reduced cell viability, colony formation and migration in HCC
and CCA cells. Upon transduction of Hep55.1C cells with sgRNA against Sbno1, tumors grew significantly less compared
to control guide RNA. We observed that SBNO1 knockdown induced distinct gene expression patterns in HCC and CCA
cell lines. However, BioID revealed that SBNO1 protein similarly modulates gene regulation in HCC and CCA by binding to
general transcription factors TAF4 and TAF3 and the transcriptional activator PNN. In addition, Sbno1 knockout
significantly inhibited liver tumor growth in three different mouse models using hydrodynamic tail vein injection of
Akt/NRas, Akt/MYC and Akt/N1ICD resulting in HCC, mixed HCC/CCA and CCA, respectively. Immunohistochemical
analyses revealed that Sbno1 deletion led to reduced angiogenesis in the tumor margin and biliary differentiation
indicating that Sbno1 is necessary for oncogene-driven tumor formation including Notch activation in CCA.
Conclusions: We identified SBNO1 as a new epigenetic driver required for HCC and iCCA tumor cell proliferation in vitro
and in vivo.
This research was funded by German Research Foundation SFB TRR 209 and by the German Cancer Research Aid.
Speakers
Stephanie Roessler